Abstract

Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We have
recently identified two types of cytolethal distending toxin (cdt) gene in C.
hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed a
PCR-restriction fragment length polymorphism (RFLP) assay that can differentiate
Chcdt-I from Chcdt-II. When the PCR-RFLP assay was
applied to 17 other Campylobacter strains and 25
non-Campylobacter strains, PCR products were not obtained irrespective
of their cdt gene-possession, indicating that the specificity of the
PCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C.
hyointestinalis strains including 23 analyzed in the previous study and 12
newly isolated from pigs and bovines, all of them showed the presence of
cdt genes. Furthermore, a restriction digest by EcoT14-I revealed that
29 strains contained both Chcdt-I and Chcdt-II and 6
strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly,
however, PCR products obtained from 7 C. hyointestinalis strains were not
completely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigested
PCR product was homologous to cdtB but not to Chcdt-IB
or Chcdt-IIB, indicating the presence of another cdt
gene-variant. Then, we further digested the PCR products with DdeI in addition to
EcoT14-I, showing that all three cdt genes, including a possible new
Chcdt variant, could be clearly differentiated. Thus, the PCR-RFLP
assay developed in this study is a valuable tool for evaluating the Chcdt
gene-profile of bacteria.

Keywords: cdt genes, C. hyointestinalis, PCR-RFLP

Campylobacter hyointestinalis was first identified from a pig with
proliferative enteritis [6, 8] and has recently been isolated from both diseased and healthy animals and
raw milk [4, 7,
9, 15].
Furthermore, this species was isolated from human diarrheal stool samples [5, 14, 16]. At present, C. hyointestinalis is
considered to be an emerging zoonotic pathogen, and its clinical importance and pathogenic
mechanism are under evaluation.

Cytolethal distending toxin (CDT), which is a genotoxin capable of directly damaging DNA in
target cells, is considered to be a possible virulence factor of various Gram-negative
bacteria including C. hyointestinalis. CDT consists of three
subunits, CdtA, CdtB and CdtC, which are encoded by the genes cdtA,
cdtB and cdtC, respectively [3]. The CDT holotoxin causes G2/M cell cycle arrest, cytoplasmic
distension and eventually cell death via apoptosis [3].
Although the pathogenic mechanism of CDT in vivo is not well understood, CDT
produced by Campylobacter jejuni has been reported to cause panmural
inflammation with mucosal denudation and necrosis affecting the jejunum, ileum and colon in
mice [10].

Recently, we have identified two types of cdt gene clusters in C.
hyointestinalis, namely, the cdt-I (Chcdt-I) and
cdt-II (Chcdt-II) genes from C.
hyointestinalis strains 022 [17] and
ATCC35217T [12], respectively. The toxins
encoded by these genes (ChCDT-I and ChCDT-II, respectively) both induced cell distention and
death in HeLa cells. However, the homologies between these two ChCDTs were only 25.0, 56.0 and
24.8% in their CdtA, CdtB and CdtC subunits, respectively. Since there was a low homology
between ChCDT-I and ChCDT-II, particularly regarding their CdtA and CdtC subunits, which are
responsible for binding to receptor molecules, it is possible that their target cells might
differ. Thus, ChCDT-I and ChCDT-II may have different pathogenic mechanisms in
vivo. Therefore, it is important to analyze the distribution of these two
cdt gene-variants in C. hyointestinalis to understand the
difference in pathogenesis between ChCDT-I and ChCDT-II. In this study, we have developed a
PCR-restriction fragment length polymorphism (PCR-RFLP) assay for the detection and
differentiation of Chcdt-I and Chcdt-II in C.
hyointestinalis. The specificity and sensitivity of the PCR-RFLP assay were
evaluated, and the presence and types of cdt genes in 35 C.
hyointestinalis strains, including 12 strains newly isolated from pigs and bovines,
were successfully determined by the PCR-RFLP assay.

MATERIALS AND METHODS

Bacterial strains and growth media

Thirty-five strains of C. hyointestinalis, which are described in Table 1, were examined in this study. Among them, 23 strains were known to possess
Chcdt-I and/or Chcdt-II [12], and the other 12 strains were newly isolated from pigs and
bovines. A total of 42 bacterial strains other than C. hyointestinalis
including 17 strains of 11 other Campylobacter species and 25 strains of
20 non-Campylobacter species were also included in this study (Table 1).

DNA preparation

Template DNAs for PCR were prepared by the boiling method as previously reported [1, 11]. Briefly,
bacterial colonies grown on appropriate agar plates were suspended in 500
µl of TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM
ethylenediaminetetraacetic acid). Bacterial suspensions were boiled for 10 min, kept on
ice for 10 min and centrifuged at 20,000 ×g for 10 min. Then, the
supernatants were collected and used as DNA templates for PCR. As positive controls for
the PCR and RFLP assays, pET28a Chcdt-IB and pET28a
Chcdt-IIB, which carry the Chcdt-IB and
Chcdt-IIB genes of C. hyointestinalis strains 022 and
ATCC35217T, respectively, were used [12, 17].

PCR amplification assay

PCR was performed with ChCdt-BF (5′-GCTACTTGGAATATGCAAGG-3′) and ChCdt-BR
(5′-TGGTTCTCTATTRAAATCWCC-3′) primer set using an Applied Biosystems Veriti®
Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Each PCR mixture contained
0.5 and 0.15 µM of ChCdt-BF and ChCdt-RF primers, respectively, 1
µl of DNA template, 0.2 mM of each dNTP, 1 × rTaq DNA
polymerase buffer and 0.5 U of rTaq DNA polymerase (Takara Bio Inc.,
Otsu, Japan) in a total volume of 20 µl. The samples were subjected to an
initial denaturation step at 94°C for 3 min followed by 30 cycles of amplification, each
cycle consisting of 94°C for 30 sec, 54°C for 30 sec and 72°C for 30 sec. A final
extension step at 72°C for 3 min was included. PCR products were analyzed by
electrophoresis using a 2% PrimeGel™ Agarose LE gel (Takara Bio Inc.), and bands were
visualized with ultraviolet (UV) light after staining with ethidium bromide (1
µg/ml). Images were captured on a ChemiDoc system
(Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.).

RFLP assay

PCR products (2 to 5 µl; 100 to 200 ng) were digested
with either 4 U of EcoT14-I or 2 U of EcoT14-I and 2 U of DdeI (New England Biolabs Inc.,
Ipswich, MA, U.S.A.) under 1 ×H buffer or 1 ×K buffer (Takara Bio Inc.) in a final volume
of 10 µl at 37°C overnight. Then, the digested PCR products were analyzed
by 3% PrimeGel™ Agarose LE gel electrophoresis as described above.

Detection limit of the PCR-RFLP assay

The C. hyointestinalis strains, ATCC35217T, 022 and 3197,
were cultured on blood agar under anaerobic conditions at 37°C for 2 days, and the
colonies that grew were suspended in sterile phosphate-buffered saline. The density of
each bacterial culture was adjusted to OD600=1.0 and then 10-fold serially
diluted in phosphate-buffered saline, and DNA templates were prepared from 100
µl of each dilution by the boiling method as described above. Then, PCR
assays were carried out with these DNA templates. Furthermore, to determine the viable
bacterial count, 100 µl of each dilution was spread on blood agar plates
in triplicate and cultured at 37°C for 2 days under anaerobic conditions, and then, the
number of colonies on each plate was counted.

Colony hybridization assay

The distribution of Chcdt-IB and Chcdt-IIB in 12 newly
isolated strains of C. hyointestinalis was examined by colony
hybridization assay as described previously [11],
with minor modiﬁcations. In brief, Chcdt-IB and
Chcdt-IIB gene-probes were prepared by PCR with each primer set (Table
S1) using C. hyointestinalis strains 022 (Chcdt-IB) and
ATCC35217T(Chcdt-IIB) as DNA templates, and the obtained PCR
products were purified from agarose gel using the Wizard SV® Gel and PCR
clean-up system (Promega Corporation, Madison, WI, U.S.A.). Strains were grown on a
nitrocellulose membrane (GE Healthcare U.K. Ltd., Buckinghamshire, U.K.) overlaid on blood
agar plates under anaerobic conditions at 37°C overnight. Colonies were lysed, and DNA was
denatured in situ by the alkaline lysis method followed by UV
cross-linking with a UV crosslinker (CX-2000; UVP LLC, Upland, CA, U.S.A.). The processed
nitrocellulose membranes were hybridized with each gene probe, and radioactivity was
visualized using an FLA-7000 biomolecular imager (GE Healthcare U.K. Ltd.).

Sequencing of cdt genes and PCR products

To determine the entire nucleotide sequence of the cdt gene cluster in
12 newly obtained C. hyointestinalis strains (Table 1), PCR was performed with a combination of forward
primers targeting upstream of cdtA or cdtB and reverse
primers targeting downstream of cdtC (Table S2). Each PCR mixture
contained 0.5 µM of each primer, 1 µl of DNA template,
0.2 mM of each dNTP, 1 ×Ex Taq® DNA polymerase buffer and 1.0
U of Ex Taq® DNA polymerase in a total volume of 20
µl. The samples were subjected to an initial denaturation step of 3 min
at 94°C followed by 30 cycles of 94°C for 30 sec, 50°C for 30 sec and 72°C for 90 sec to 2
min, and then a final extension step at 72°C for 3 to 5 min. If a PCR product of the
expected size was obtained, the PCR product was purified using a PCR clean-up system
(QIAGEN GmbH, Hilden, Germany) for sequencing. The sequencing reactions were performed by
the chain termination method with the BigDye® Terminator v1.1 cycle sequencing
kit (Thermo Fisher Scientific). Nucleotide sequences were determined using an ABI
PRISM® 3130-Avant Genetic analyzer (Thermo Fisher
Scientific). The sequence upstream of Chcdt-IB in C.
hyointestinalis strains 130206DCC11, 130206DCC12, 130325D2aC1, 141007D1C1,
S2TDNEFB-1, S3C-1 and S5C-1, and that of Chcdt-IIB in C.
hyointestinalis strain 141007D2C1, were determined with the primers listed in
Table S2 by genome walking as described previously [12].

Undigested PCR products (approximately 500 bp in length) were purified from agarose gel
by using the Wizard® SV Gel and PCR clean-up system (Promega Corporation). The
nucleotide sequences of undigested PCR products were determined as described above,
analyzed using MEGA6, and compared with the sequences of Chcdt-IB and
Chcdt-IIB.

Nucleotide sequence accession numbers

The cdt gene-sequences determined in this study were deposited to the
DNA Data Bank of Japan (DDBJ) with the accession numbers LC175774 to LC175800.

RESULTS

Evaluation of ChcdtB amplification by PCR

By comparing published cdtB sequences of C.
hyointestinalis (GenBank accession nos. AB218983 and AB373951) and those of
other Campylobacter spp. (AB274783, AB274793, AB274802, AB872889 and
AB872911), forward and reverse primers (ChCdt-BF and ChCdt-BR) were designed from the
conserved regions of Chcdt-IB and Chcdt-IIB. To evaluate
whether these primers can amplify Chcdt-IB and
Chcdt-IIB, PCR was carried out using recombinant plasmids, pET28a
carrying Chcdt-IB and pET28a carrying Chcdt-IIB, as
positive controls. PCR of the positive controls, pET28a carrying Chcdt-IB
and pET28a carrying Chcdt-IIB, gave the expected size of the PCR product
(507 or 516 bp) in each case (Fig. S1). Sequence analysis further confirmed that these PCR
products were amplified from Chcdt-IB and Chcdt-IIB
gene-fragments (data not shown).

The sensitivity of the PCR assay was evaluated with the 35 C.
hyointestinalis strains listed in Table 1. A PCR product of the expected size was obtained from 23 strains, which
were previously reported to possess Chcdt-I and/or
Chcdt-II (Table 1).
Furthermore, a PCR product of the same size was also obtained from 12 newly isolated
C. hyointestinalis strains in which the presence of
Chcdt-I and/or Chcdt-II was confirmed by colony
hybridization assay and sequence analysis as described below. Then, the specificity of the
PCR assay was evaluated with a total of 42 strains including 17 strains of 11 other
Campylobacter spp. and 25 strains of 20
non-Campylobacter spp. as listed in Table 1. Irrespective of their cdt
gene-possession, no PCR product was obtained from those 42 strains.

To calculate the sensitivity of the PCR-RFLP assay developed in this study, we further
evaluated the distribution of cdt genes in 12 C.
hyointestinalis strains, which were newly isolated from pigs and bovines, by
colony hybridization assay. The Chcdt-IB gene-probe was reacted with 8
strains (130206DCC11, 130206DCC12, 130325D2aC1, 141007D1C1, 141007D2C1, S2TDNEFB-1, S3C-1
and S5C-1), while the Chcdt-IIB gene-probe was reacted with all the
strains (data not shown). Furthermore, the entire nucleotide sequences of these
cdt gene clusters were determined as described in the Materials and
Methods section. The sizes of individual cdt genes and the homology of
each cdt gene are summarized in Table S3. These data indicated that the
12 C. hyointestinalis strains possessed Chcdt-I and/or
Chcdt-II, except for 1 strain, 141007D2C1, in which
cdtA was missing.

A ChcdtB-based PCR-RFLP assay

EcoT14-I restriction enzyme was selected for the PCR-RFLP assay by comparing the
sequences of Chcdt-IB and Chcdt-IIB. When the PCR
products obtained from the positive controls were digested with EcoT14-I, the
Chcdt-IB DNA fragment yielded two bands of 363 and 144 bp (Fig. 1A, lane 1), and in the case of Chcdt-IIB, the fragment sizes were
261 and 255 bp (Fig. 1A, lane 2). We then
evaluated the utility of the PCR-RFLP assay with the PCR products obtained from 35
C. hyointestinalis strains (Table 1). Among the 35 strains, the
cdt gene-possession profile of 23 strains was previously analyzed by
colony hybridization assay and the same types of cdt were successfully
identified by their RFLP patterns, even though both Chcdt-I and
Chcdt-II were present (Fig.
1A, lane 4). When the PCR products obtained from 12 other strains were analyzed
by the RFLP assay, their RFLP patterns indicated that all C.
hyointestinalis strains contained Chcdt-IIB and 7 strains
additionally possessed Chcdt-IB (Table 1). These data indicated that the PCR-RFLP assay could identify the
cdt gene-profile. However, undigested PCR products were obtained from 7
strains (3197, 3535, 3839, 3857, 87–4, 130206DCC11 and 141007D2C1) among 35 strains when
digested with EcoT14-I (Fig. 1A, lane 5).

(A) PCR-RFLP with EcoT14-I to evaluate the presence of the ChcdtB
gene in C. hyointestinalis. PCR products digested with EcoT14-I
were analyzed by electrophoresis on a 3% agarose gel. Undigested PCR product in lane
5 was extracted from the gel, purified,...

The undigested PCR products obtained from 7 strains were purified and sequenced. This
analysis revealed that the undigested PCR products carried sequences of
cdt genes with only 45–46% and 64–66% homologies to the corresponding
regions of Chcdt-IB and Chcdt-IIB, respectively. These
data indicated that the C. hyointestinalis strains might possess new
variants of cdt genes.

To avoid the possibility of obtaining ambiguous results from the PCR-RFLP assay, we
further digested the PCR products with DdeI in addition to EcoT14-I. When the PCR products
obtained from positive controls were digested with these enzymes, the
Chcdt-IB gene-fragment and Chcdt-IIB gene-fragment
were theoretically digested to yield fragments of 363, 101 and 43 bp, and 261, 113, 70, 41
and 31 bp, respectively. The agarose gel separation result is shown in Fig. 1B. Furthermore, the PCR products of the
cdtB gene-homologue were theoretically digested to yield fragments of
302, 72, 51, 27 and 24 bp (Fig. 1B). Then, when
we evaluated the utility of this modified PCR-RFLP assay with the PCR products obtained
from 35 C. hyointestinalis strains, Chcdt-IB,
Chcdt-IIB and cdtB gene-homologue could be clearly
distinguished from one another.

DISCUSSION

Although the clinical significance of C. hyointestinalis infection has not
yet been clearly established, C. hyointestinalis is implicated as a
pathogen of both humans and animals. We have previously reported that two types of the
cdt genes, namely Chcdt-I and Chcdt-II,
are present in C. hyointestinalis and encode biologically active CDTs
[12, 17].
Therefore, CDT is a candidate virulence factor in C. hyointestinalis. To
understand the importance of CDT in the pathogenesis of C. hyointestinalis,
we attempted to establish a PCR-RFLP assay for the detection and differentiation of
Chcdt-I and Chcdt-II in C.
hyointestinalis. Since cdtB was previously demonstrated to be
more conserved than cdtA and cdtC in C.
jejuni, C. coli and C. fetus [2], various cdtB gene-based multiplex PCR
and PCR-RFLP assays have been developed to detect and differentiate
Campylobacter species [1, 11, 13]. In this
study, we have developed a ChcdtB-based PCR-RFLP assay that can detect and
differentiate between Chcdt-I and Chcdt-II.

The sensitivity and specificity of the PCR-RFLP assay were shown to be 100%. Furthermore,
the PCR-RFLP assay could clearly identify the cdt gene-profile, even though
C. hyointestinalis contained both Chcdt-I and
Chcdt-II (Fig. 1A, lane 4).
Kamei et al. have reported that Chcdt-II may be
ubiquitously conserved in C. hyointestinalis [12]. In this study, we evaluated the presence of cdt
genes using 12 C. hyointestinalis strains that were newly isolated from
pigs and bovines. As expected, Chcdt-IIB was detected in all the C.
hyointestinalis strains, while Chcdt-IB was also detected in 7
strains (Table 1). Furthermore, the presence of
Chcdt-IIA and Chcdt-IIC in these 12 strains was analyzed
by colony hybridization assay, and their nucleotide sequences were determined by sequence
analysis (Table S3). Although Chcdt-IIA was not detected in C.
hyointestinalis strain 141007D2C1, 11 other strains were demonstrated to possess
Chcdt-IIA, Chcdt-IIB and Chcdt-IIC. It
appears that Chcdt-IIB and Chcdt-IIC are ubiquitously
present in C. hyointestinalis and may be suitable target genes for the
detection and identification of this species.

The isolation of C. hyointestinalis is difficult if C.
jejuni and C. coli are targeted for isolation because of its
susceptibility to cephem antibiotics, which are included in the modified
charcoal-cefoperazone-deoxycholate agar and Bolton broth that are normally used for the
isolation of C. jejuni and C. coli. Therefore, a PCR-based
method is required to detect this bacterium before initiating cultivation. The
cdtB gene-based PCR assay developed in this study could detect C.
hyointestinalis with 100% sensitivity and specificity (Table 1). The detection limit of the assay was determined to be
approximately 102 CFU/20 µl of reaction mixture. These data
indicate that the PCR assay developed in this study is useful for the detection of
C. hyointestinalis.

However, PCR products obtained from some template DNAs of C.
hyointestinalis strains remained undigested by EcoT14-I (Fig. 1A, lane 5). A sequence analysis of these PCR fragments showed
that these strains carry the homologous cdtB gene sequence, indicating the
detection of a new cdt gene-variant of C. hyointestinalis.
Therefore, we further included DdeI digestion in the cdtB gene-based
PCR-RFLP assay to distinguish not only Chcdt-IB and
Chcdt-IIB but also a possible new cdtB gene-variant. The
cdtB gene-based PCR-RFLP assay with EcoT14-I and DdeI could clearly
distinguish three different cdtB genes as PCR products including
Chcdt-IB and Chcdt-IIB and a possible new
cdtB gene-variant in the 35 C. hyointestinalis strains
listed in Table 1. Further analysis to
characterize the cdt gene-variant is currently ongoing in our
laboratory.

In conclusion, the ChcdtB gene-based PCR-RFLP assay developed in this
study is useful to detect and differentiate not only Chcdt-I and
Chcdt-II but also a possible new cdt gene-variant in
C. hyointestinalis. The ubiquitous presence of Chcdt-IIB
among the tested C. hyointestinalis strains also confirmed it to be an
appropriate target gene for the identification of C. hyointestinalis.
Further studies are required for the evaluation of the cdtB gene-based
PCR-RFLP assay for the detection and differentiation of cdt genes present
in C. hyointestinalis.

Supplementary Material

Acknowledgments

We thank Dr. Rupak K. Bhadra (CSIR-Indian Institute of Chemical Biology,
Kolkata, India) for the critical reading of the manuscript. This study was performed in
partial fulfillment of the requirements of a Ph.D. thesis for N. Hatanaka from the Graduate
School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.